More than 80% of global transmission of HIV infection occurs via mucosal routes. The ability of vaccines to induce mucosal immunity may be required for protection against HIV infection and the immunodeficiency syndrome that emerges after infection. Various AIDS vaccine candidates are currently under development, each with potential advantages and disadvantages. DNA vaccines have features that make them attractive vaccine candidates for HIV and the combination of DNA vaccination plus recombinant vaccinia is one of the favored approaches in AIDS vaccine development. We have established a vaccination regimen that consistently stimulates mucosal responses with an SIV DNA vaccine, which consists of a single plasmid carrying an SIV proviral genome that produces noninfectious particles. We have the appropriate tools to measure SlV-specific IgA levels in mucosal secretions, mucosal cell-mediated responses, and SlV-specific systemic responses. As a consequence, we believe that we are uniquely placed to establish the effectiveness of mucosal DNA vaccination and how stimulation of virusspecific mucosal responses can affect the efficacy of a HIV/SIV vaccine. This proposal is designed to extend our previous studies by investigating approaches to maximize immune responses to DNA vaccination in primates using a SIV construct that produces non infectious virus and has been previously tested in Rhesus macaques. Toward this goal we propose: 1. To evaluate whether the combination of ovalbumin and LT in Eldexomer administered nasally or vaginally to Rhesus macaques stimulates significant anti-ovalbumin IgA and possibly IgG titers in vaginal and rectal secretions. 2. To evaluate: a. whether the addition of a vaginal gp130 Env protein boosting immunization to the nasal or intramuscular (i.m.) SIV DNA-rMVA regimen leads to higher levels of Env-specific IgA titers at mucosal membranes that are sites of HIV entry in most human infections; b. how these titers compare to what we have achieved with combined intradermal (i.d.) plus mucosal immunization. 3. To evaluate the different homing receptor characteristics of SlV-specific cells induced via different immunization routes and determine which route is better suited for inducing virus specific responses that home at mucosal membranes that are sites of HIV entry in humans.